Developing a new treatment for superficial fungal infection using antifungal Collagen‐HSAF dressing

Abstract Fungal pathogens are common causes of superficial clinical infection. Their increasing drug resistance gradually makes existing antifungal drugs ineffective. Heat stable antifungal factor (HSAF) is a novel antifungal natural product with a unique structure. However, the application of HSAF has been hampered by very low yield in the current microbial producers and from extremely poor solubility in water and common solvents. In this study, we developed an effective mode of treatment applying HSAF to superficial fungal infections. The marine‐derived Lysobacter enzymogenes YC36 contains the HSAF biosynthetic gene cluster, which we activated by the interspecific signaling molecule indole. An efficient extraction strategy was used to significantly improve the purity to 95.3%. Scanning electron microscopy images revealed that the Type I collagen‐based HSAF (Col‐HSAF) has a transparent appearance and good physical properties, and the in vitro sustained‐release effect of HSAF was maintained for more than 2 weeks. The effective therapeutic concentration of Col‐HSAF against superficial fungal infection was explored, and Col‐HSAF showed good biocompatibility, lower clinical scores, mild histological changes, and antifungal capabilities in animals with Aspergillus fumigatus keratitis and cutaneous candidiasis. In conclusion, Col‐HSAF is an antifungal reagent with significant clinical value in the treatment of superficial fungal infections.


| INTRODUCTION
Fungi infect billions of people every year, yet the contribution of fungi to the global burden of disease is largely unrecognized. Although true mortality rates are unknown because of a lack of good epidemiological data, the incidence of invasive fungal infections is rising as a result of modern medical interventions and immunosuppressive diseases. 1 Of the estimated 5 million or more fungal species worldwide, approximately 300 fungal species have been documented as causing human disease, but only 20-25 do so on a relatively frequent basis, including the fungal species A. fumigatus and Candida krusei. 2 Compared with antibacterial research, the development of new antifungal drugs has been relatively slow, and there are only four major classes of antifungal agents (polyenes, flucytosine, azoles, and echinocandins). 3 For example, among the clinically common antifungal agents, amphotericin B (AMB) and natamycin (E235) belong to the polyene class which can destroy fungal cell walls, while voriconazole is the azole class that can exert an antifungal effect by inhibiting ergosterol production. A number of hurdles to antifungal drug development must be overcome, one of which is the problem of co-toxicity for eukaryotic targets: as fungi are eukaryotic pathogens with a number of important similarities with their human hosts, antifungals often exhibit unacceptably high toxicity to human cells, which complicates the search for antifungal targets and slows the development of antifungal agents. 4 Over the past 5-6 years, with the widespread use of antifungal agents, there has been a clear increase in the identification of yeasts that have developed resistance. 3 Therefore, promising new antifungal agents are urgently needed to meet clinical needs.
HSAF isolated from L. enzymogenes strains has exhibited broadspectrum antifungal activity. [5][6][7] Previously, HSAF was used for the biological control of plant fungal diseases by interfering in the biosynthesis of fungal sphingolipids, a mechanism that differs from that of other antifungal drugs. 8 Its chemical structure contains a tetramic acid moiety and a 5,5,6-tricyclic skeleton, which is different from any existing antifungal drug. Due to this complex structure, it is extremely difficult to obtain HSAF through organic chemical synthesis. At present, HSAF can be produced only by Lysobacter, and its biosynthetic gene cluster and synthesis mechanism have been gradually revealed. 6,7 The biosynthetic genes of HSAF include a single module of PKS/NRPS, arginase, 5 redox enzymes, and sterol desaturation/fatty acid hydroxylase. [9][10][11] To date, HSAF has not been synthesized by chemical means, and L. enzymogenes is one of the few strains that can produce HSAF. The regulatory mechanism of HSAF biosynthesis and how to improve the yield of HSAF are still largely unknown. HSAF has been used in only agricultural fungal diseases since its discovery. As HSAF has a novel chemical structure and antifungal mechanism, solving the shortcomings of low yield, inadequate purity, and low solubility would greatly increase HSAF's therapeutic value and broaden its application scope.
In combination with its relatively poor solubility even in organic solvents, the cytotoxicity of HSAF in organic solvents has prevented its extension into the clinic. 12 The discovery and development of new drugs are not sufficient to achieve therapeutic excellence and enter the drug market. The topical delivery of antifungal drugs is perhaps the best treatment for superficial fungal infections; its advantages include site-specific drug delivery, decreased systemic toxicity, increased efficacy of treatment, and improved bioavailability. 13 Conventional formulations require high dosages and repeated administration, associated with an increased risk of both local and systemic toxicity. A new drug delivery system for insoluble HSAF is necessary to reduce local side effects and increase therapeutic efficacy. 14 Hydrogels are promising materials for drug delivery and wound healing applications due to their good biodegradability, low toxicity, and well-known safety profile. 15 On skin contact, these formulations form a semi-occlusive film over the skin and release the drug in a controlled manner, which is ideal for the topical delivery of HSAF. 16 In this study, we identified a novel strategy for the treatment of superficial fungal infections with HSAF. We optimized an HSAF purification technology by using marine-derived L. enzymogenes YC36, in which HSAF production was induced by indole, a ubiquitous interspe-  and Pseudomonas aeruginosa PAO1. Its inhibitory level against grampositive bacteria, such as Bacillus subtilis 168, is weak (Figure 1a). We explored the biological mechanism by analyzing the genome of L. enzymogenes YC36, which revealed 15 secondary metabolite biosynthetic gene clusters, including the antifungal compound HSAF ( Figure 1b). We investigated the production of HSAF in L. enzymogenes YC36 with bacterial growth. HSAF starts to be produced in the lag phase, but production is very low in the first 36 h and peaks from 36-48 h, followed by a gradual decrease in production ( Figure 1c). In the stationary phase, the expression of PKS/NRPS in L. enzymogenes YC36 increased significantly (Figure 1d). In addition, 2% NaCl promoted the expression of PKS/NRPS and the production of HSAF (Figure 1e,f), probably because 2% NaCl is most closely to the original growth environment of L. enzymogenes YC36. The efficient and high-purity extraction of HSAF is one of the bottlenecks restricting its subsequent research. The HSAF purification process is shown in Figure 2a as a schematic diagram. Initially, the purity of HSAF in the supernatant of bacterial liquid was 59.28% according to high-performance liquid chromatography (HPLC) data. The microporous resin was used to adsorb HSAF in the supernatant. The adsorption rate was >95%, and residual HSAF was rarely detected in the culture medium ( Figure S1). Subsequently, after eluting the impurities from the microporous resin with low to high methanol concentrations, the purity of HSAF collected in 100% methanol increased to 68.46%, and the recovery was 70.55%. HPLC is the final step in HSAF purification, the recovery rate of HPLC was 52.48% (Figure 2b), and the ultimate purity of HSAF increased to 95.3% ( Figures S2 and S3).

| Stimulation of HSAF production by indole
As mentioned above, the low yield of HSAF is one of the bottlenecks affecting its subsequent application. In this study, we were surprised to find that indole, a signaling molecule, significantly improved the expression of approximately two-thirds of the biosynthesis gene clusters of secondary metabolites in L. enzymogenes YC36 (Figure 3a). It is worth noting that the expression of the HSAF gene cluster was upregulated by 9-fold, while the expression of the WAP-8294A2 gene cluster was upregulated by 10-fold. The HSAF biosynthesis gene cluster contains 10 orfs. They include major facilitator superfamily transporters (orf1), alcohol dehydrogenase zinc-binding protein (orf2), F I G U R E 1 L. enzymogenes YC36 inhibits the growth of bacteria and fungi. (a) Effects of L. enzymogenes YC36 on the growth of selected fungi and gram-negative and gram-positive bacteria. Fungi include F. solani ATCC 36031, A. niger ATCC 16404, A. niger CMCC 98003 (f), A. fumigatus AS 3.1320, and C. krusei ATCC 14243. Gram-negative bacteria include E. coli ATCC 25923 and P. aeruginosa PAO1, and gram-positive bacteria include B. subtilis 168. (b) Analysis of 15 secondary metabolite gene clusters in L. enzymogenes YC36. (c) The growth and peak area of HSAF in L. enzymogenes YC36 at 0-72 h. The lag phase: 0-6 h, the exponential phase: 6-36 h, the stationary phase: 36-60 h, and the decline phase: 60-72 h. (d) The relative expression levels of PKS/NRPS at different phases in L. enzymogenes YC36. The lag phase was selected as the control experiment. (e) The relative expression levels of PKS/NRPS in L. enzymogenes YC36 cultured with different concentrations of NaCl. The 0% NaCl concentration was selected as the control for the experiment. (f) The effect of different NaCl concentrations on HSAF production in L. enzymogenes YC36. The 0% NaCl concentration was selected as the control for the experiment. NS, not significant, *p < 0.05, **p < 0.01, and ***p < 0.001, replicates = 3. All data are presented as means ± SEM, and statistical significance was determined using paired t tests Three strains of bacteria were selected as the control conditions. The results show that the antifungal effect of HSAF was much stronger than the antibacterial effect. NS, not significant, *p <0.05, **p <0.01, and ***p <0.001, replicates =3. All data are presented as means ± SEM, and statistical significance was determined using paired t tests 500 μM indole increased production by approximately 1.5-fold ( Figure 3d). Therefore, 200 μM indole was selected for subsequent experiments to increase HSAF production and did not affect the growth speed of L. enzymogenes YC36 (Figure 3e). The two-component system QseC/ QseB was shown to be sensitive to indoles. 17 If qseC/qseB was knocked out, PKS/NRPS expression was significantly downregulated, regardless of the presence or absence of indole ( Figure 3f). In addition, the combination of 2% NaCl and 200 μM indole exerted a greater effect on improving the HSAF yield than the addition of a single molecule, which is important for future studies on increasing HSAF production ( Figure 3g).

| Synthesis of Col-HSAF and property characterization
HSAF is insoluble in water and is difficult to use clinically as a topical antifungal agent for its biotoxicity, as shown in our preliminary experiment ( Figure S4). To solve this problem, we chose Type I collagen as . NS, not significant, *p < 0.05, **p < 0.01, and ***p < 0.001, n =5 samples per group. All data are presented as means ± SEM, and statistical significance was determined using paired t tests and C. krusei (Table 1; Figure S6). A transmission electron micrograph showed that the density of fungal hyphae was obviously richer and the length was longer in the Ctrl group than in the Col-HSAF group, and fewer spores were observed in the Col-HSAF group (Figure 5e).

| Therapeutic effects of Col-HSAF on A. fumigatus keratitis and cutaneous candidiasis
Mouse models of two fungal infections were constructed to further evaluate the inhibitory effects of Col-HSAF on fungal proliferation in vivo: A. fumigatus keratitis (FK) and cutaneous candidiasis.
In addition, a fungal loading test (Figure 6c)  T A B L E 2 Primary pharmacokinetic parameters of Col-HSAF in rabbit eyes   . NS, not significant, *p < 0.05, **p < 0.01, and ***p < 0.001, n =5 animals per group. All data are presented as means ± SEM, and statistical significance was determined using paired t tests Previously, HSAF was isolated from two terrestrial strains, L. enzymogenes C3 and OH11. 6,7 Although there have been many published studies on the biosynthetic mechanism of HSAF, there are relatively few studies on its regulation and bioengineering. Strains capable of producing HSAF is limited, so it is important to explore new strains capable of producing HSAF in special environments. In this study, we found that L. enzymogenes YC36 isolated from marine environments can inhibit the growth of many fungi and bacteria by producing abundant secondary metabolites. The whole genome of L. enzymogenes YC36 contains 15 biosynthetic gene clusters of secondary metabolites, including the antifungal compound HSAF. 18,19 In contrast to other terrestrial HSAF-producing bacteria, in L. enzymogenes YC36, HSAF production is improved by regulating the NaCl concentration in nutrient-deprived environments.
In addition, the majority of HSAF is lost during purification because of its poor solubility. A previous study showed that HSAF production is increased by 17.95% using a two-stage temperature control strategy and adding glucose improve the yield of HSAF. 8,20 Moreover, the deletion of c-di-GMP genes from L. enzymogenes OH11 improves the yield of HSAF. 21 In this study, we found a new method to increase the expression of the HSAF gene cluster and the yield of HSAF by adding the exogenous interspecific signaling molecule indole. Previously, indole has been widely studied in bacterial resistance and other bacterial social activities. 17,22 However, there are relatively few studies on the effects of secondary metabolite production. Here, we proved that an indole-mediated increase in HSAF production is regulated by the two-component system QseC/QseB.
HSAF is a highly potent antifungal compound, but it is cytotoxic and poorly soluble in water. In the wider drug release literature, release from hydrogel polymers is a popular and safe approach. 23,24 Therefore, we chose to use a Type I collagen membrane as the HSAF carrier, which combines good biocompatibility with high light transmittance and mechanical strength, ensuring that it will not significantly affect vision after being attached to the ocular surface.
Collagen is a fibrous protein found in all multicellular animals and serves as a major constituent of many connective tissues. The administration of collagen to patients has also been reported to be safe and has been used as a biomaterial for antifungal drug delivery. 25,26 Sustained release of HSAF from Col-HSAF was observed. On the one hand, HSAF was insoluble in water and contains hydrophobic groups in its molecular structure. It might interact with the hydrophobic groups on the collagen chain such as tyrosine, phenylalanine, and tryptophan during the process of collagen film formation, and this molecule was not easy to dissolve in the water again in an aqueous solution. On the other hand, after the collagen was dried and a film formed, the structure of Col-HSAF was dense and the swelling rate in water was limited. The HSAF on the surface layer was released fast, while the HSAF on the inner layer was wrapped in the layered collagen and was difficult to diffuse. The antifungal activity of HSAF has been investigated in a previous study 5

| Strains and general methods
The strains used in this study are shown in Table S1. L. enzymogenes strains and the derived mutants were grown in LB medium and 40% TSB medium, and the optimal incubation temperature was 28 C.   Then the dissolved Vor was diluted to 10 μg/ml with sterile normal saline.

| Extraction of HSAF
Sulfobutylether-β-cyclodextrin was dissolved in water at a concentration of 2 mg/ml. Then, the HSAF solution was slowly added dropwise to the sulfobutylether-β-cyclodextrin solution at a mass ratio of HSAF to cyclodextrin of 3:10, and the mixture was stirred at room temperature for 2 h and allowed to stand for 30 min. Next, the solution was filtered with a 0.45 μm microporous membrane, and sulfobutylether-β-cyclodextrin-HSAF was obtained after freezedrying.
For the preparation of the HSAF-lanolin ointment, lanolin and the HSAF solution was mixed at a mass ratio of lanolin:HSAF = 1000:1.

| Inhibition zone test
The previously prepared AFSP was shaken in a test tube; then, 300 μl

| Clinical scoring
The inoculated eyes were scored with a slit lamp at 1, 3, 5, and 7 days.
A grade of 0 to 4 was assigned to the area of opacity, density of opacity, and surface regularity. The area of opacity was graded as follows: The mice were sacrificed on the 3rd day postinfection, and the cornea was penetratively cut from the eyeball and the iris was removed; the cornea was cut and spread radially on a glass slide, 100 g/L KOH was Guangdong Hengjian Co., Ltd) was applied once daily to Group 4; and Groups 5 and 6 was treated with lanolin or the Type I hydrogel without drug, respectively. The specific usage is described below. In the Col-HSAF and Col groups, the Col-HSAF and Col membranes were cut into fragments using a trephine with a diameter of 2.5 mm 2 and attached to the skin of mice; membranes were replaced once daily. In the clotrimazole group, the drug was smeared on the skin surface once daily. In the Ctrl group, PBS was smeared on the skin surface once daily.

| C. krusei CFUs
Swabs were collected from each infected area and placed in sterile tubes containing 5 ml of yeast malt agar during 7 days of treatment.
Serial dilutions were prepared and then 1 ml of each dilution was inoculated into Petri dishes containing 10 ml of PDA. The inoculated plates were incubated for 24 h at 37 C, and the colonies were counted.

| Histopathological examination
On the 7th day postinfection, after taking skin swabs, the mice were sacrificed, and the tested part of the shaved skin was excised and fixed in 10% buffered formalin. The fixed skin tissues were processed and embedded in paraffin. Sections were cut from the paraffin block by microtome and stained with hematoxylin and eosin. The structure was observed under a light microscope (Leica, DM-6000, Wetzlar, Germany) with an integrating camera to identify histopathological changes.

| Statistical analysis
The statistical analyses were performed using SPSS version 21.0 (IBM). Differences in the clinical scores and fungal viability results were analyzed via one-way ANOVA with the least significant difference post hoc test. Significant differences were defined as a p value <0.05.
In the pharmacokinetics study, noncompartmental pharmacokinetic parameters were calculated using the WinNonLin 8.

| Data availability
All data associated with this study are present in the paper or the Supplementary Materials.

| CONCLUSIONS
In this study, we developed an effective mode of treatment by applying HSAF to superficial fungal infections. The marine-derived

CONFLICT OF INTEREST
The authors declare no competing interests.

DATA AVAILABILITY STATEMENT
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.